User:Cody Couperus/Sandbox 1: Difference between revisions
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* Activation of [http://www.ncbi.nlm.nih.gov/pubmed/17008302 thrombin activatable fibrinolysis inhibitor (TAFI)] <ref>PMID: 20229688</ref> | * Activation of [http://www.ncbi.nlm.nih.gov/pubmed/17008302 thrombin activatable fibrinolysis inhibitor (TAFI)] <ref>PMID: 20229688</ref> | ||
Activity of thrombin is regulated physiologically by the serpin inhibitors: | Activity of thrombin is regulated physiologically by the [http://en.wikipedia.org/wiki/Serpin serpin inhibitors]: | ||
* [[Antithrombin]] <ref name="three"/><ref name='four'>PMID: 24477356</ref><ref name='five'>PMID: 23809129</ref> | * [[Antithrombin]] <ref name="three"/><ref name='four'>PMID: 24477356</ref><ref name='five'>PMID: 23809129</ref> | ||
* [http://en.wikipedia.org/wiki/Heparin_cofactor_II Heparin cofactor II]<ref name="three"/><ref name='four'/><ref name='five'/> | * [http://en.wikipedia.org/wiki/Heparin_cofactor_II Heparin cofactor II]<ref name="three"/><ref name='four'/><ref name='five'/> | ||
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Thrombin interacts with platelet membrane protein [http://www.ncbi.nlm.nih.gov/pubmed/14720584 GpIbα] and subsequently cleave protease activated receptor-1 (PAR1) causing a signaling cascade which leads to platelet [http://en.wikipedia.org/wiki/Platelet_alpha-granule α-granule] release and membrane flipping exposing the negatively charged [http://en.wikipedia.org/wiki/Phosphatidylserine phosphatidylserine]. The platelet alpha granules contain the physiologically relevant pool of FVa. <ref>Camire, R. M. (2010). Platelet factor V to the rescue. Blood, 115(4), 753-754. [http://bloodjournal.hematologylibrary.org/content/115/4/753.full DOI: 10.1182/blood-2009-11-252619]</ref> | Thrombin interacts with platelet membrane protein [http://www.ncbi.nlm.nih.gov/pubmed/14720584 GpIbα] and subsequently cleave protease activated receptor-1 (PAR1) causing a signaling cascade which leads to platelet [http://en.wikipedia.org/wiki/Platelet_alpha-granule α-granule] release and membrane flipping exposing the negatively charged [http://en.wikipedia.org/wiki/Phosphatidylserine phosphatidylserine]. The platelet alpha granules contain the physiologically relevant pool of FVa. <ref>Camire, R. M. (2010). Platelet factor V to the rescue. Blood, 115(4), 753-754. [http://bloodjournal.hematologylibrary.org/content/115/4/753.full DOI: 10.1182/blood-2009-11-252619]</ref> | ||
Thrombin also causes activation of FIX, through | Thrombin also causes activation of FIX, through [http://en.wikipedia.org/wiki/Factor_XI FX] cleavage, and FVIII which form the Xase complex to activate FX. FVa and FXa form the [http://en.wikipedia.org/wiki/Prothrombinase prothrombinase] complex in the presence of calcium and phospholipid. It causes rapid activation of prothrombin to thrombin. This increase in thrombin allows sufficient fibrinogen to be cleaved to fibrin which is able to polymerize to form a stable [http://en.wikipedia.org/wiki/Thrombus blood clot]. | ||
Further supporting coagulation, thrombin activates FXIII, a transglutaminase that crosslinks fibrin at lysine residues | Further supporting coagulation, thrombin activates FXIII, a [http://en.wikipedia.org/wiki/Transglutaminase transglutaminase] that crosslinks fibrin at lysine residues. | ||
Once the endothelial lining is reached thrombin binds heparin glycosaminoglycans. This facilitates its inactivation by serpin inhibitors antithrombin and heparin cofactor II. In addition, thrombin will interact with thrombomodulin which significantly increases its catalytic efficiency activating protein C. Activated protein C inactivates FVIIa and FVa thus down regulating thrombin generation. | The structure of thrombin facilitates its inactivation. Once the endothelial lining is reached thrombin binds [http://en.wikipedia.org/wiki/Heparin heparin] [http://en.wikipedia.org/wiki/Glycosaminoglycan glycosaminoglycans]. This facilitates its inactivation by serpin inhibitors antithrombin and heparin cofactor II. In addition, thrombin will interact with [http://en.wikipedia.org/wiki/Thrombomodulin thrombomodulin] which significantly increases its catalytic efficiency activating protein C. Activated protein C inactivates FVIIa and FVa thus down regulating thrombin generation. | ||
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Prothrombin is the zymogen form of thrombin. From N-terminal to C-terminal it consists of a Gla domain, two kringle domains, and a catalytic domain. | Prothrombin is the zymogen form of thrombin. From N-terminal to C-terminal it consists of a Gla domain, two kringle domains, and a catalytic domain. | ||
Prothrombin is activated by prothrombinase which consists of FXa, FVa, calcium, and a phospholipid surface. In vivo the first cleavage occurs at the R320-I321 bond, corresponding to residues 15-16 in thrombin which is the N-terminus of the B chain, producing meizothrombin.<ref name='seven'>PMID: 22944689</ref> Subsequent cleavage at R271-T272 yields thrombin.<ref name='seven'/> The initial cleavage can also occur at R271 resulting in | Prothrombin is activated by prothrombinase which consists of FXa, FVa, calcium, and a phospholipid surface. In vivo the first cleavage occurs at the R320-I321 bond, corresponding to residues 15-16 in thrombin which is the N-terminus of the B chain, producing meizothrombin.<ref name='seven'>PMID: 22944689</ref> Subsequent cleavage at R271-T272 yields thrombin.<ref name='seven'/> The initial cleavage can also occur at R271 resulting in prethrombin-2 which will then be cleaved at R320 to produce thrombin.<ref>PMID: 1995649</ref> | ||
After cleavage by prothrombinase the new B chain N-terminus (Ile16) folds into the core protease domain and forms a salt bridge with Asp194.<ref name='seven'/> This leads to stabilization of regions of the 180s-loop, Na+ binding loop, and γ-loop (zymogen activation domains). These changes provide the correct conformation for the S1 pocket and oxyanion hole for catalysis.<ref name='seven'/><ref>PMID: 15890651</ref> | After cleavage by prothrombinase the new B chain N-terminus (Ile16) folds into the core protease domain and forms a salt bridge with Asp194.<ref name='seven'/> This leads to stabilization of regions of the 180s-loop, Na+ binding loop, and γ-loop (zymogen activation domains). These changes provide the correct conformation for the S1 pocket and oxyanion hole for catalysis.<ref name='seven'/><ref>PMID: 15890651</ref> | ||
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[[Image:Electrostatic labeled.png|300px|right|thumb| Thrombin (1PPB) overlayed with electrostatic surface. Structural features 60-loop, γ-loop, exosite I, and exosite II labeled]] | [[Image:Electrostatic labeled.png|300px|right|thumb| Thrombin (1PPB) overlayed with electrostatic surface. Structural features 60-loop, γ-loop, exosite I, and exosite II labeled]] | ||
Thrombin is a α/β heterodimer composed of a 36 amino acid A chain and 259 amino acid B chain connected by a <scene name='58/583418/Disulfides_nospin/1'>disufide</scene> bridge between Cys1 and Cys122, in addition to 3 other intrachain disulfide bonds.<ref name='eight'>PMID: 2583108</ref> Its overall fold is similar to trypsin and chymotrypsin and it belongs to the peptidase S1 protease family<ref>PMID: 18768474</ref>. It is an overall spherical protein with approximate dimensions of 45 X 45 X 50 Å | Thrombin is a α/β heterodimer composed of a 36 amino acid A chain and 259 amino acid B chain connected by a <scene name='58/583418/Disulfides_nospin/1'>disufide</scene> bridge between Cys1 and Cys122, in addition to 3 other intrachain disulfide bonds.<ref name='eight'>PMID: 2583108</ref> Its overall fold is similar to trypsin and chymotrypsin and it belongs to the [http://merops.sanger.ac.uk/cgi-bin/famsum?family=s1 peptidase S1 protease family]<ref>PMID: 18768474</ref>. It is an overall spherical protein with approximate dimensions of 45 Å X 45 Å X 50 Å.<ref name='eight'/> | ||
Important structural features include: | Important structural features include: | ||
* A prominent active site cleft flanked by the 60- and γ-loops | * A prominent active site cleft flanked by the 60- and γ-loops | ||
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* Two surface patches referred to as exosite I and exosite II. | * Two surface patches referred to as exosite I and exosite II. | ||
The <scene name='58/583418/A_chain_nospin/1' target='0'>A chain</scene> is mostly helical and is wound around the B chain and shaped like a boomerang. It is bound to the B chain mostly through side chain interactions including a salt bridge and | The <scene name='58/583418/A_chain_nospin/1' target='0'>A chain</scene> is mostly helical and is wound around the B chain and shaped like a boomerang. It is bound to the B chain mostly through side chain interactions including a salt bridge and a hydrogen bond cluster at residues D14, E8, and E14c.<ref name='eight'/> Furthermore the C-terminus region forms a short amphipathic helix with hydrophobic side chains interacting with the B chain.<ref name='eight'/> | ||
The <scene name='58/583418/B_chain/1' target='0'>B chain</scene> contains the active site of the protein and has numerous notable structural features. The active site is formed at the rims of two interacting 6 stranded <scene name='58/583418/Beta_barrel/2'>beta barrel domains</scene>(N-terminal barrel in red and C-terminal barrel in orange) which are surrounded by 4 helical regions and many turns. | The <scene name='58/583418/B_chain/1' target='0'>B chain</scene> contains the active site of the protein and has numerous notable structural features. The active site is formed at the rims of two interacting 6 stranded <scene name='58/583418/Beta_barrel/2'>beta barrel domains</scene>(N-terminal barrel in red and C-terminal barrel in orange) which are surrounded by 4 helical regions and many turns. | ||
The serine protease <scene name='58/583418/Catalytic_triad/1'>catalytic triad</scene>, based on chymotrypsin numbering, are Ser195, His57, and Asp102. As is common with serine proteases, an <scene name='58/583418/Oxyanion_hole/2'>oxyanion hole</scene> hole is formed by backbone amides of Ser195 and Gly193.<ref name='seven'/> This has the functional role of stabilizing the oxyanion intermediate involved in the serine protease mechanism by hydrogen bonding to the oxygen of the P1 residue ( | The serine protease <scene name='58/583418/Catalytic_triad/1'>catalytic triad</scene>[http://en.wikipedia.org/wiki/Catalytic_triad wiki] residues, based on chymotrypsin numbering, are Ser195, His57, and Asp102. As is common with serine proteases, an <scene name='58/583418/Oxyanion_hole/2'>oxyanion hole</scene> hole is formed by backbone amides of Ser195 and Gly193.<ref name='seven'/> This has the functional role of stabilizing the oxyanion intermediate involved in the serine protease mechanism by hydrogen bonding to the oxygen of the P1 residue (standard substrate-protease nomeclature <ref>PMID: 22925665</ref>. In addition, since thrombin cleaves after Arg/Lys the <scene name='58/583418/S1/2'>S1 specificity site</scene>, formed by the 180s- and 220s- loops, has Asp189 at the base to form a salt bridge with the incoming substrate. Furthermore, the S4 binding pocket accommodates hydrophobic substrate residues. | ||
The '''active site''' cleft rims are formed by the hydrophobic and rigid <scene name='58/583418/Loops/2'>60-loops</scene> (residues L60, Y60a, P60b, P60c, W60d, D60e, K60f, N60g, F60h, T60i, and N60g) and the <scene name='58/583418/Loops/1'>γ-loop</scene> (residues T147, W147a, T147b, A147c, N147d, and V147f) while the base is mostly hydrophilic negatively charged amino acids. The cleft is deep compared to more promiscuous serine proteases, consequently substrates must either have a large loop that is cleaved or have favorable interactions with the insertion loops <ref>PMID: 16102053</ref>. | The '''active site''' cleft rims are formed by the hydrophobic and rigid <scene name='58/583418/Loops/2'>60-loops</scene> (residues L60, Y60a, P60b, P60c, W60d, D60e, K60f, N60g, F60h, T60i, and N60g) and the <scene name='58/583418/Loops/1'>γ-loop</scene> (residues T147, W147a, T147b, A147c, N147d, and V147f) while the base is mostly hydrophilic negatively charged amino acids. The cleft is deep compared to more promiscuous serine proteases, consequently substrates must either have a large loop that is cleaved or have favorable interactions with the insertion loops <ref>PMID: 16102053</ref>. | ||
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'''Exosite II''' is also part of the B chain, and derived from numerous basic amino acids, this is the site of heparin binding through the sulfate groups on the glycosaminoglycan. It is also the site of GpIα on the platelet surface. | '''Exosite II''' is also part of the B chain, and derived from numerous basic amino acids, this is the site of heparin binding through the sulfate groups on the glycosaminoglycan. It is also the site of GpIα on the platelet surface. | ||
The <scene name='58/583418/Sodium_binding_loop/1'>sodium binding site</scene> is formed by the 180s- and 220s- loops. Na+ is bound by the backbone oxygens of Arg221a and Lys224 in addition to four water molecules in a classic octahedral geometry<ref>PMID: 9108691</ref>. Through the covelent disulfide linkage between Cys220 and Cys 191 the sodium binding site is linked to Ser195 and the oxyanion hole. | The <scene name='58/583418/Sodium_binding_loop/1'>sodium binding site</scene> is formed by the 180s- and 220s- loops. Na+ is bound by the backbone oxygens of Arg221a and Lys224 in addition to four water molecules in a classic [http://chemwiki.ucdavis.edu/Inorganic_Chemistry/Crystal_Field_Theory/High_Spin_and_Low_Spin_Complexes#Octahedral_Geometry octahedral geometry]<ref>PMID: 9108691</ref>. Through the covelent disulfide linkage between Cys220 and Cys 191 the sodium binding site is linked to Ser195 and the oxyanion hole. | ||
==Thrombin Allostery== | ==Thrombin Allostery== | ||